JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1976, p. 359-363 Copyright © 1976 American Society for Microbiology
Vol. 3, No. 3 Printed in U.S.A.
Inhibition of Bacteroides fragilis on Blood Agar Plates and Reversal of Inhibition by Added Hemin TRACY D. WILKINS,* SARAH L. CHALGREN, F. JIMENEZ-ULATE, C. R. DRAKE, JR., AND J. L. JOHNSON
Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received for publication 31 October 1975
Bacteroides fragilis strains formed much smaller colonies on most types of blood agar plates than they did on the same media without blood. Blood inhibited strains of B. fragilis subsp. distasonis the most and B. fragilis subsp. fragilis the least. The inhibition could be eliminated by adding hemin to the blood agar. The inhibitory component of the blood was inside the erythrocytes and appeared to be the hemin-free globin of hemoglobin. final concentration of hemin remaining irn solution was determined by using the extinction coefficient of the reduced alkaline pyridine hemochrome (2). All glassware was heated at 490 C for 4 h to destroy any residual hemin. Bacterial strains. All strains were from the culture collection of the Virginia Polytechnic Institute (VPI) Anaerobe Laboratory. All subspecies designations of B. fragilis are according to the deoxyribonucleic acid (DNA) homology groups described by Johnson (4). A strain of B. fragilis subsp. distasonis (VPI B1-20) was used for tests of activity of blood fractions. The cultures were grown overnight in chopped meat (CM) broth (3) in CO2-filled tubes. Agar well tests for inhibition. Activity of blood fractions was determined by measuring zones of inhibition around wells cut into Schaedler agar plates. The wells were 5 mm in diameter and were cut with a sterile cork borer. The surface of the plate was MATERIALS AND METHODS swabbed with a 1:1,000 dilution of an overnight culBlood. Defibrinated sheep blood was obtained ture of strain B1-20 in CM broth, and the wells were from the Brown Laboratory, Topeka, Kan. Fresh filled with the material to be tested. The plate was blood from sheep, goats, horses, rabbits, and cows incubated for 24 h in a GasPak anaerobe jar at 37 C. was collected aseptically and was defibrinated by Diameters of zones of inhibition were measured. Colony diameters. Isolated colonies were obgentle shaking with glass beads. Blood was stored at 5 C. For some experiments, the blood was laked tained either by spreading 0.1 ml of a 10-; dilution of an overnight CM culture onto agar plates or by (hemolyzed) by freezing and thawing. Media. Media for plates were made aerobically streaking cultures directly onto plates. Isolated coland sterilized at 121 C for 15 min. Media were ob- onies were measured with a dissection microscope tained from BBL, Baltimore, Md., except for brain equipped with an ocular micrometer. Five to ten heart infusion (BHI) agar (Difco, Detroit, Mich.) to isolated colonies on each plate were measured, and which yeast extract (Difco) was added to give a final averages were reported. Fractionation of blood. Defibrinated sheep blood concentration of 0.5% in the medium. Hemin solutions. For routine tests, we used (50 ml) was centrifuged for 10 min at 755 x g, and hemin dissolved in NaOH (3), but for the more criti- the erythrocytes were washed four times with 0.85% cal experiments reported in Table 1 and Fig. 2 we NaCl. After the final wash, the cells were lysed by used the following alkaline-ethanolic solution A. suspending them in 250 ml of distilled water. The Solution A was prepared by dissolving 200 mg of lysed cells were incubated with ribonuclease (12 jig/ hemin (Sigma, St. Louis, Mo., equine type III) in 10 ml, bovine pancreatic type A, Sigma) for 30 min at ml of 0.2 M KOH in 47.5% ethanol, adding 40 ml of room temperature, and the cell stroma were rewater, and stirring at maximum speed with a mag- moved from the lysate by centrifuging it two times netic stirrer for 2 h. This solution was filtered for 30 min at 16,300 x g. The supernatant was then through membrane filters (Millipore Corp., Bedford, diluted to 1 liter with 0.02 M phosphate buffer (pH Mass.) of 0.8-, 0.65-, 0.4-, and 0.22-gm pore size. The 7.5). 359
During the course of experiments to determine which agar medium would best support growth of anaerobic bacteria, we found that Bacteroides fragilis strains grew better on agar medium without blood than on the same medium with 5% blood added. With some strains, there was complete inhibition of growth on the blood plates. Because of the clinical importance of B. fragilis and the common use of blood agar plates in clinical laboratories, we have investigated this phenomenon in greater detail. This communication reports that inhibition is dependent on the medium used, and describes the degree of inhibition of the different subspecies of B. fragilis and the reversal of the inhibitory activity of blood by added hemin.
360
WILKINS ET AL.
J. CLIN. MICROBIOL.
Columbia blood plates. There was no apparent inhibition on brucella blood agar. The oxidation-reduction potential of the media did not seem to be a factor, because inhibition was observed with prereduced media in roll tubes as well as with aerobically prepared agar plates. The extent of inhibition varied with the subspecies (DNA homology group) of B. fragilis tested. B. fragilis subsp. distasonis was inhibited the most, and B. fragilis subsp. fragilis was inhibited the least (Table 2). Although there were strain variations within each homology group, none of the B. fragilis subsp. fragilis strains was inhibited as much as any of the B. fragilis subsp. distasonis strains. The other subspecies were intermediate in extent of inhibition (Table 2). The type of animal blood used to make the blood agar plates also had an effect on the degree of inhibition. Blood from either sheep or cows was the most inhibitory, and rabbit blood was the least inhibitory (Table 3). Blood from different shipments and different animals also varied in the extent of inhibition. Reversal of inhibition by added hemin. Hemin is a required growth factor for all B. fragilis subspecies, and it is routinely added to media for growing this organism. Because blood contains large amounts of hemin, it is often not added to blood agar plates; however, the addition of hemin to BHI blood plates eliminated the inhibiting effect of blood. There was a direct dose response in colony size to the amount of hemin added to the medium (Fig. 2). Considerably more hemin had to be added to the BHI blood agar than to the regular BHI agar to obtain the same size colonies, and with RESULTS laked (lysed) blood plates, even larger amounts The inhibitory activity of blood for B. fragilis of hemin had to be added to obtain maximum strains was demonstrated by swabbing the sur- growth (Fig. 2). Schaedler agar necessitated the face of agar plates with a sensitive strain and addition of the largest amount of hemin to elimfilling wells in the plates with whole blood. inate the inhibition. Although this medium Zones of inhibition appeared, and with many of contains 10 ,ug of hemin per ml when made the sensitive strains there was a complete ab- from the commercial mixture, an additional 4 sence of growth near the blood (Fig. 1). The ,g/ml had to be added to obtain maximum inhibition was also obvious when cultures were colony size with whole blood, and 40 Ag/ml was streaked on blood agar plates. Some sensitive necessary with laked blood. This medium supstrains did not form isolated colonies on BHI ported good growth in the absence of blood, blood agar plates after 3 days of incubation, indicating that the hemin was available for whereas colonies of 2-mm diameter occurred growth. after overnight incubation on plain BHI plates. Isolation of inhibitory component. The acFactors affecting extent of inhibition. The tivity of blood fractions was measured by deterextent of inhibition was dependent upon the mining the diameter of zones of inhibition type of agar medium used for the blood agar around wells in Schaedler agar plates. With plates (Table 1). Colonies of B. fragilis subsp. this assay, we found that the inhibitory activity distasonis B1-20 were smallest on Schaedler of whole blood was primarily inside the erythroand BHI blood agar plates, and intermediate in cytes. The inhibitory activity was retained size on Trypticase soy, blood agar base, and after the erythrocytes were washed with saline.
Solid ammonium sulfate was added to the solution at room temperature (22 C) to achieve successive concentrations of 50, 70, 80, and 100% saturation. The solution was allowed to sit for 30 min at each saturation level, and the precipitate was then removed by centrifuging for 30 min at 16,300 x g. The precipitates were dissolved in the 0.02 M phosphate buffer, dialyzed overnight against several changes of 2 liters of the same buffer, diluted to 50 ml with the buffer, and filter sterilized. These ammonium sulfate fractions were assayed for activity by the agar well method. The material resulting from the 80% (NH4)2SO4 saturation was further fractionated on a Sephadex G-100 column and on an isoelectric focusing column (LKB-Produkter AB; 8100-20). Two milliliters of the sample was applied to a Sephadex column (2.5 by 32 cm) that had been equilibrated with 0.03 M phosphate buffer at pH 7.0. Another 1 ml of the sample was fractionated on the isoelectric focusing column filled with a linear 0 to 50% sucrose gradient containing a pH 5 to 8 ampholyte (LKB) solution. The sample was incorporated in the gradient, and a voltage of 1,000 V was applied to the column for 48 h at 2 C. Isoelectric focusing concentrates each molecule at its isoelectric point; a detailed description of the method and apparatus has been written by Vesterberg (6). The column was emptied through a flow cell on a Gilford 2400 spectrophotometer, and fractions were assayed for activity with the agar well test. Hemoglobin and native globin. Two-times-recrystallized sheep and bovine hemoglobins were obtained from Sigma. Hemin was removed from the sheep hemoglobin by extraction at 5 C with acidacetone (5). The precipitate was washed with acetone, dissolved in water, and slowly titrated over a 2-h period with NaOH to neutrality (5). This material was tested for activity.
VOL. 3, 1976
INHIBITION OF B. FRAGILIS ON BLOOD AGAR
361
FIG. 1. Inhibition of B. fragilis subsp. distasonis strain B1-20 by whole sheep blood contained in a 5-mmdiameter well cut in a BHI agar plate supplemented with 5 pg of hemin per ml.
When the erythrocytes were lysed with distilled water, the cell membranes were not inhibitory, but the supernatant was. The activity in the supernatant was not destroyed by heating at 56 C for 30 min, but was eliminated by heating
at 80 C for 10 min. Chocolate blood agar plates also were not inhibitory.
The inhibitory activity of the supernatant was retained by a 10,000-molecular-weight cutoff ultrafilter (Amicon, PM-10), was nondialyz-
362
WILKINS ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Colony diameters of B. fragilis subsp. distasonis strain Bl-20 on six media with and without 5% sheep blood Colony diam (mm) No blood
Medium Blood
No
E 2.0
E
u-
1.5
>_
1.0
Hemin
hemin
(10
j±g/ml)
BHI 0-0.1 0 1.9 Schaedler -O_.la 2.0' 2.0a Trypticase soy 0.3 0 1.2 Columbia 0.5 0 1.9 Blood agar base 0.6 0 1.0 Brucella 1.1 0 1.3 a Schaedler medium contains 10 /ig of hemin per ml in the commercial mixture that was used for this experiment. Hemin concentrations refer to the amount of additional hemin added. TABLE 2. Colony diameters of five DNA homology groups (subspecies) of B. fragilis on BHI agar with and without sheep blood
z
0 0 () 0.5 (9 cr
I 0 ) 0.25 0.50 1.0 2.0 4.0 8.0 16.0 32.0
AMOUNT OF HEME ADDED (p.g/mf) FIG. 2. Colony diameters of strain Bl-20 on BHI plates and BHI sheep blood plates as a function of amount of added hemin.
Colony diam (mm)a B. fragilis subsp.
BHI + blood
BHI + hemin
fragilis 1.0 + 0.5 1.9 ± 0.5 ovatus 0.5 ± 0.2 1.9 ± 0.6 thetaiotaomicron 0.3 ± 0.1 1.6 ± 0.3 vulgatus 0.3 ± 0.1 1.7 ± 0.3 distasonis 0.1 + 0.1 1.5 ± 0.4 a Colony diameters ± standard deviation, after 48 h of incubation. Average of 10 strains was calculated, except 9 strains were used with B. fragilis subsp. vulgatus. TABLE 3. Colony diameters of strain B1-20 on BHI blood agar plates made with blood from several species Type of blood
Sheep Cow Horse Goat Rabbit None + 5 ,ug of hemin per ml
Colony diam (mm)
Vol. 3, No. 3 Printed in U.S.A.
Inhibition of Bacteroides fragilis on Blood Agar Plates and Reversal of Inhibition by Added Hemin TRACY D. WILKINS,* SARAH L. CHALGREN, F. JIMENEZ-ULATE, C. R. DRAKE, JR., AND J. L. JOHNSON
Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received for publication 31 October 1975
Bacteroides fragilis strains formed much smaller colonies on most types of blood agar plates than they did on the same media without blood. Blood inhibited strains of B. fragilis subsp. distasonis the most and B. fragilis subsp. fragilis the least. The inhibition could be eliminated by adding hemin to the blood agar. The inhibitory component of the blood was inside the erythrocytes and appeared to be the hemin-free globin of hemoglobin. final concentration of hemin remaining irn solution was determined by using the extinction coefficient of the reduced alkaline pyridine hemochrome (2). All glassware was heated at 490 C for 4 h to destroy any residual hemin. Bacterial strains. All strains were from the culture collection of the Virginia Polytechnic Institute (VPI) Anaerobe Laboratory. All subspecies designations of B. fragilis are according to the deoxyribonucleic acid (DNA) homology groups described by Johnson (4). A strain of B. fragilis subsp. distasonis (VPI B1-20) was used for tests of activity of blood fractions. The cultures were grown overnight in chopped meat (CM) broth (3) in CO2-filled tubes. Agar well tests for inhibition. Activity of blood fractions was determined by measuring zones of inhibition around wells cut into Schaedler agar plates. The wells were 5 mm in diameter and were cut with a sterile cork borer. The surface of the plate was MATERIALS AND METHODS swabbed with a 1:1,000 dilution of an overnight culBlood. Defibrinated sheep blood was obtained ture of strain B1-20 in CM broth, and the wells were from the Brown Laboratory, Topeka, Kan. Fresh filled with the material to be tested. The plate was blood from sheep, goats, horses, rabbits, and cows incubated for 24 h in a GasPak anaerobe jar at 37 C. was collected aseptically and was defibrinated by Diameters of zones of inhibition were measured. Colony diameters. Isolated colonies were obgentle shaking with glass beads. Blood was stored at 5 C. For some experiments, the blood was laked tained either by spreading 0.1 ml of a 10-; dilution of an overnight CM culture onto agar plates or by (hemolyzed) by freezing and thawing. Media. Media for plates were made aerobically streaking cultures directly onto plates. Isolated coland sterilized at 121 C for 15 min. Media were ob- onies were measured with a dissection microscope tained from BBL, Baltimore, Md., except for brain equipped with an ocular micrometer. Five to ten heart infusion (BHI) agar (Difco, Detroit, Mich.) to isolated colonies on each plate were measured, and which yeast extract (Difco) was added to give a final averages were reported. Fractionation of blood. Defibrinated sheep blood concentration of 0.5% in the medium. Hemin solutions. For routine tests, we used (50 ml) was centrifuged for 10 min at 755 x g, and hemin dissolved in NaOH (3), but for the more criti- the erythrocytes were washed four times with 0.85% cal experiments reported in Table 1 and Fig. 2 we NaCl. After the final wash, the cells were lysed by used the following alkaline-ethanolic solution A. suspending them in 250 ml of distilled water. The Solution A was prepared by dissolving 200 mg of lysed cells were incubated with ribonuclease (12 jig/ hemin (Sigma, St. Louis, Mo., equine type III) in 10 ml, bovine pancreatic type A, Sigma) for 30 min at ml of 0.2 M KOH in 47.5% ethanol, adding 40 ml of room temperature, and the cell stroma were rewater, and stirring at maximum speed with a mag- moved from the lysate by centrifuging it two times netic stirrer for 2 h. This solution was filtered for 30 min at 16,300 x g. The supernatant was then through membrane filters (Millipore Corp., Bedford, diluted to 1 liter with 0.02 M phosphate buffer (pH Mass.) of 0.8-, 0.65-, 0.4-, and 0.22-gm pore size. The 7.5). 359
During the course of experiments to determine which agar medium would best support growth of anaerobic bacteria, we found that Bacteroides fragilis strains grew better on agar medium without blood than on the same medium with 5% blood added. With some strains, there was complete inhibition of growth on the blood plates. Because of the clinical importance of B. fragilis and the common use of blood agar plates in clinical laboratories, we have investigated this phenomenon in greater detail. This communication reports that inhibition is dependent on the medium used, and describes the degree of inhibition of the different subspecies of B. fragilis and the reversal of the inhibitory activity of blood by added hemin.
360
WILKINS ET AL.
J. CLIN. MICROBIOL.
Columbia blood plates. There was no apparent inhibition on brucella blood agar. The oxidation-reduction potential of the media did not seem to be a factor, because inhibition was observed with prereduced media in roll tubes as well as with aerobically prepared agar plates. The extent of inhibition varied with the subspecies (DNA homology group) of B. fragilis tested. B. fragilis subsp. distasonis was inhibited the most, and B. fragilis subsp. fragilis was inhibited the least (Table 2). Although there were strain variations within each homology group, none of the B. fragilis subsp. fragilis strains was inhibited as much as any of the B. fragilis subsp. distasonis strains. The other subspecies were intermediate in extent of inhibition (Table 2). The type of animal blood used to make the blood agar plates also had an effect on the degree of inhibition. Blood from either sheep or cows was the most inhibitory, and rabbit blood was the least inhibitory (Table 3). Blood from different shipments and different animals also varied in the extent of inhibition. Reversal of inhibition by added hemin. Hemin is a required growth factor for all B. fragilis subspecies, and it is routinely added to media for growing this organism. Because blood contains large amounts of hemin, it is often not added to blood agar plates; however, the addition of hemin to BHI blood plates eliminated the inhibiting effect of blood. There was a direct dose response in colony size to the amount of hemin added to the medium (Fig. 2). Considerably more hemin had to be added to the BHI blood agar than to the regular BHI agar to obtain the same size colonies, and with RESULTS laked (lysed) blood plates, even larger amounts The inhibitory activity of blood for B. fragilis of hemin had to be added to obtain maximum strains was demonstrated by swabbing the sur- growth (Fig. 2). Schaedler agar necessitated the face of agar plates with a sensitive strain and addition of the largest amount of hemin to elimfilling wells in the plates with whole blood. inate the inhibition. Although this medium Zones of inhibition appeared, and with many of contains 10 ,ug of hemin per ml when made the sensitive strains there was a complete ab- from the commercial mixture, an additional 4 sence of growth near the blood (Fig. 1). The ,g/ml had to be added to obtain maximum inhibition was also obvious when cultures were colony size with whole blood, and 40 Ag/ml was streaked on blood agar plates. Some sensitive necessary with laked blood. This medium supstrains did not form isolated colonies on BHI ported good growth in the absence of blood, blood agar plates after 3 days of incubation, indicating that the hemin was available for whereas colonies of 2-mm diameter occurred growth. after overnight incubation on plain BHI plates. Isolation of inhibitory component. The acFactors affecting extent of inhibition. The tivity of blood fractions was measured by deterextent of inhibition was dependent upon the mining the diameter of zones of inhibition type of agar medium used for the blood agar around wells in Schaedler agar plates. With plates (Table 1). Colonies of B. fragilis subsp. this assay, we found that the inhibitory activity distasonis B1-20 were smallest on Schaedler of whole blood was primarily inside the erythroand BHI blood agar plates, and intermediate in cytes. The inhibitory activity was retained size on Trypticase soy, blood agar base, and after the erythrocytes were washed with saline.
Solid ammonium sulfate was added to the solution at room temperature (22 C) to achieve successive concentrations of 50, 70, 80, and 100% saturation. The solution was allowed to sit for 30 min at each saturation level, and the precipitate was then removed by centrifuging for 30 min at 16,300 x g. The precipitates were dissolved in the 0.02 M phosphate buffer, dialyzed overnight against several changes of 2 liters of the same buffer, diluted to 50 ml with the buffer, and filter sterilized. These ammonium sulfate fractions were assayed for activity by the agar well method. The material resulting from the 80% (NH4)2SO4 saturation was further fractionated on a Sephadex G-100 column and on an isoelectric focusing column (LKB-Produkter AB; 8100-20). Two milliliters of the sample was applied to a Sephadex column (2.5 by 32 cm) that had been equilibrated with 0.03 M phosphate buffer at pH 7.0. Another 1 ml of the sample was fractionated on the isoelectric focusing column filled with a linear 0 to 50% sucrose gradient containing a pH 5 to 8 ampholyte (LKB) solution. The sample was incorporated in the gradient, and a voltage of 1,000 V was applied to the column for 48 h at 2 C. Isoelectric focusing concentrates each molecule at its isoelectric point; a detailed description of the method and apparatus has been written by Vesterberg (6). The column was emptied through a flow cell on a Gilford 2400 spectrophotometer, and fractions were assayed for activity with the agar well test. Hemoglobin and native globin. Two-times-recrystallized sheep and bovine hemoglobins were obtained from Sigma. Hemin was removed from the sheep hemoglobin by extraction at 5 C with acidacetone (5). The precipitate was washed with acetone, dissolved in water, and slowly titrated over a 2-h period with NaOH to neutrality (5). This material was tested for activity.
VOL. 3, 1976
INHIBITION OF B. FRAGILIS ON BLOOD AGAR
361
FIG. 1. Inhibition of B. fragilis subsp. distasonis strain B1-20 by whole sheep blood contained in a 5-mmdiameter well cut in a BHI agar plate supplemented with 5 pg of hemin per ml.
When the erythrocytes were lysed with distilled water, the cell membranes were not inhibitory, but the supernatant was. The activity in the supernatant was not destroyed by heating at 56 C for 30 min, but was eliminated by heating
at 80 C for 10 min. Chocolate blood agar plates also were not inhibitory.
The inhibitory activity of the supernatant was retained by a 10,000-molecular-weight cutoff ultrafilter (Amicon, PM-10), was nondialyz-
362
WILKINS ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Colony diameters of B. fragilis subsp. distasonis strain Bl-20 on six media with and without 5% sheep blood Colony diam (mm) No blood
Medium Blood
No
E 2.0
E
u-
1.5
>_
1.0
Hemin
hemin
(10
j±g/ml)
BHI 0-0.1 0 1.9 Schaedler -O_.la 2.0' 2.0a Trypticase soy 0.3 0 1.2 Columbia 0.5 0 1.9 Blood agar base 0.6 0 1.0 Brucella 1.1 0 1.3 a Schaedler medium contains 10 /ig of hemin per ml in the commercial mixture that was used for this experiment. Hemin concentrations refer to the amount of additional hemin added. TABLE 2. Colony diameters of five DNA homology groups (subspecies) of B. fragilis on BHI agar with and without sheep blood
z
0 0 () 0.5 (9 cr
I 0 ) 0.25 0.50 1.0 2.0 4.0 8.0 16.0 32.0
AMOUNT OF HEME ADDED (p.g/mf) FIG. 2. Colony diameters of strain Bl-20 on BHI plates and BHI sheep blood plates as a function of amount of added hemin.
Colony diam (mm)a B. fragilis subsp.
BHI + blood
BHI + hemin
fragilis 1.0 + 0.5 1.9 ± 0.5 ovatus 0.5 ± 0.2 1.9 ± 0.6 thetaiotaomicron 0.3 ± 0.1 1.6 ± 0.3 vulgatus 0.3 ± 0.1 1.7 ± 0.3 distasonis 0.1 + 0.1 1.5 ± 0.4 a Colony diameters ± standard deviation, after 48 h of incubation. Average of 10 strains was calculated, except 9 strains were used with B. fragilis subsp. vulgatus. TABLE 3. Colony diameters of strain B1-20 on BHI blood agar plates made with blood from several species Type of blood
Sheep Cow Horse Goat Rabbit None + 5 ,ug of hemin per ml
Colony diam (mm)
